I. Megakaryocytopolesis
In the bone marrow pluripotent stem cells differentiate into megakaryocytic, erythrocytic, and myelocytic cell lines. It is believed there is a line of committed cells between stem cells and megakaryocytes. The earliest recognizable member of the megakaryocyte (meg) family are the megakaryoblasts. These cells are initially 20 to 30 .mu.m in diameter having basophilic cyloplasm and a slightly irregular nucleus with loose, somewhat reticular chromatin and several nucleoli. Later, megakaryoblasts may contain up to 32 nuclei, but the cytoplasm remains sparse and immature. As maturation proceeds, the nucleus becomes more lobulate and pyknotic, the cytoplasm increases in quantity and becomes more acidophilic and granular. The most mature cells of this family may give the appearance of releasing platelets at their periphery. Normally, less than 10% of megakaryocytes are in the blast stage and more than 50% are mature. Arbitrary morphologic classifications commonly applied to the megakaryocyte series: are megakaryoblast for the earliest form; promegakaryocyle or basophilic megakaryocyte for the intermediate form; and mature (acidophilic, granular, or platelet-producing) megakaryocyte for the late forms. The mature megakaryocyte extends filaments of cytoplasm into sinusoidal spaces where they detach and fragment into individual platelets (Williams et al., Hematology, 1972).
Megakaryocytopoiesis is believed to involve several regulatory factors (Williams et al., Br. J. Haematol., 52:173 [1982] and Williams et al., J. Cell Physiol. 110:101 [1982]). The early level of megakaryocytopoiesis is postulated as being mitotic, concerned with cell proliferation and colony initiation from CFU-meg but is not affected by platelet count (Burstein et al., J. Cell Physiol. 109:333 [1981] and Kimura et al., Exp. Hernatol. 13:1048 [1985]). The later stage of maturation is non-mitotic, involved with nuclear polyploidization and cytoplasmic maturation and is probably regulated in a feedback mechanism by peripheral platelet number (Odell et al., Blood 48:765 [1976] and Ebbe et al., Blood 32:787 [1968]). The existence of a distinct and specific megakaryocyte colony-stimulating factor (meg-CSF) is still in dispute (Mazur, E., Exp. Hematol. 15:340-350 [1987]). Although meg-CSF's have been partly purified from experimentally produced thrombocytopenia (Hill et al., Exp. Hematol. 14:752 [1986]) and human embryonic kidney conditioned medium [CM] (McDonald et al., J. Lab. Clin. Med. 85:59 [1975]) and in man from aplastic anemia and idiopathic thrombocylopenic purpura urinary extracts (Kawakita et al., Blood6:556 [1983]) and plasma (Hoffman et al., J. Clin. Invest. 75:1174 [1985]), their physiological function is as yet unknown in most cases. The conditioned medium of pokeweed mitogen-activated spleen cells (PWM-SpCM) and the murine myelomonocyte cell line WEHI-3 (WEHI-3CM) have been used as megakaryocyte potentiators. PWM-SpCM contains factors enhancing CFU-meg growth (Metcalf et al., Pro. Natl. Acad. Sci., USA 72:1744-1748 [1975]; Quesenberry et al., Blood 65:214 [1985]; and Iscove, N. N., in Hematopoietic Cell Differentiation, ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. 10, Golde et al., eds. [New York, Academy Press] pp 37-52 [1978]), one of which is interleukin-3 (IL-3), a multilineage colony stimulating factor (multi-CSF [Burstein, S. A., Blood Cells 11:469 [1986]). The other factors in this medium have not yet been identified and isolated. WEHI-3 is a mudne myelomonocytic cell line secreting: relatively large amounts of IL-3 and smaller amounts of GM-CSF. IL-3 has been purified and cloned (Ihle et al., J. Immunol. 129:2431 [1982]) and has been found to potentlate the growth of a wide range of hemopoietic cells (Ihle et al., J. Immunol. 13:282 [1983]). IL-3 has also been found to synergize with many of the known hemopoietic hormones or growth factors (Bartelmez et al., J. Cell Physiol. 22:362-369 [1985] and Warren et al., Cell 46:667-674 [1988]), including both erythropoietin (EPO) and H-1 (later known as interleukin-1 or IL-1), in the induction of very early multipotential precursors and the formation of very large mixed hemopoietic colonies.
Other sources of megakaryocyte potentiators have been found in the conditioned media of mudne lung, bone, macrophage cell lines, peritoneal exudate cells and human embryonic kidney cells. Despite certain conflicting data (Mazur, E., Exp. Hematol. 15:340-350 [1987]), there is some evidence (Geissler et al., Br. J. Haematol. 60:233-238 [1985]) that activated T lymphocytes rather than monocytes play an enhancing role in megakaryocytopoiesis. These findings suggest that activated T-lymphocyte secretions such as interleukins may be regulatory factors in meg development (Geissler et al., Exp. Hernatol. 15:845-853 [1987]). A number of studies on megakaryocytopoiesis with purified EPO (Vainchenker et al., Blood 54:940 [1979]; McLeod et al., Nature 261:492-4 [1976]; and Williams et al., Exp. Hematol. 12:734 [1984]) indicate that this hormone has an enhancing effect on meg colony formation. More recently this has been demonstrated in both serum-free and serum- containing cultures and in the absence of accessory cells (Williams et al., Exp. Hematol. 12:734 [1984]). EPO was postulated to be involved more in the single and two-cell stage aspects of megakaryocytopoiesis as opposed to the effect of PWM-SpCM which was involved in the four-cell stage of megakaryocyte development. The interaction of all these factors on both early and late phases of megakaryocyte development remains to be elucidated.
Other documents of interest include: Eppstein et al., U.S. Pat. No. 4,962,091; Chong, U.S. Pat. No. 4,879,111; Fernandes et al., U.S. Pat. No. 4,604,377; Wissler et al., U.S. Pat. No. 4,512,971; Gottlieb, U.S. Pat. No. 4,468,379; Kimura et al., Eur. J. Immunol., 20(9): 1927-1931 (1990); Secer, W. E. et al., J. of Immunol., 144(4):1484-1489 (1990); Warren, D. J., et al., J. of Immunol., 140(1): 94-99 (1988); Warren, M. K. et al.,, Exp. Hematol., 17(11): 1095-1099 (1989); Bruno, E., et al.,, Exp. Hematol., 17(10): 1038-1043 (1989); Tanikawa et al.,, Exp. Hematol., 17(8): 883-888 (1989); Koike et al., Blood, 75(12): 2286-2291 (1990); Lotem, et al., Blood, 75(5): 1545-1551 (1989); Rennick, D., et al., Blood, 73(7): 1828-1835 (1989); and Clutterbuck, E. J., et al., Blood, 73(6): 1504-1512 (1989)